This invention relates to the field of viral inactivation of blood products and blood product compositions, including blood, blood components, blood plasma or any fraction, concentrate or derivative thereof containing blood proteins, plasma-containing products and plasma fraction-containing products containing labile proteins, for example immunoglobulins, through use of a solvent/detergent process to accomplish same. In particular the solvents used include the di- and tri-alkyl phosphates and the detergents include partial esters of sorbital anhydrides, including oxyethylated alkylphenols and in particular the Tritons®. The blood products are thereby rendered substantially free of enveloped viruses such as for example the hepatitis viruses and other viral infectivity, such blood products and blood product compositions are thus purified.
The solvent-detergent (S/D) process has been in use for close to 20 years to inactivate enveloped viruses in plasma products; it continues to be the viral inactivation method by which other novel methods are compared. Concentrations of 1.0% detergent and 0.3% tri-n-butyl phosphate (TNBP) solvent have been considered necessary for robust removal of viral activity. The S/D treatment generally requires more chemicals and takes a longer time to perform when accomplished at the front end of the process (where the process volumes are greater and the product less well defined, as a result of raw material being typically of less purity and potency) than later in the process, when impurities have been reduced and the product is in most cases better defined and of greater purity and potency with reduced volume, or where viral load has been reduced or diminished by at least one robust viral removal step (i.e., resulting in a log removal of ≧/=4 logs for enveloped virus and ≧/=3 logs for non-enveloped virus).
This invention discloses the effectiveness of solvent-detergent treatment after fractionation and nanofiltration or size exclusion filtration of a blood product or blood product composition for example such as an immune gamma globulin preparation, allowing use of significantly reduced concentrations of solvent and detergent. This invention further discloses the surprising finding that when used after size exclusion filtration in a purified protein system, 10 to 20 times less S/D chemicals are required to accomplish complete inactivation of enveloped virus as compared to S/D viral inactivation concentrations used by fractionators and experts trained in the art over the last 20 years.
A preferred method of the instant invention discloses removal of the S/D chemicals. In accordance therewith, it is disclosed that the S/D chemicals can be effectively removed by using a diffusion column containing silica beads in which the pore volume is filled with a three-dimensional cross-linked hydrophobic acrylic polymer to reduce protein binding to the silica. Such column is specifically designed for removal of S/D from a well-defined protein solution. By practicing the solvent/detergent process at the reduced concentrations discussed herein it becomes feasible to require 10 to 20 times less column material to rid the product of solvent/detergent post the viral inactivation treatment. Such small packing makes it feasible in most cases to dispose of the chromatographical material post each use. This is important to control possible cross contamination between batches due to the presence of live non-enveloped virus and prion particles associated with TSEs.
The S/D process has continued to be the more favorable viral inactivation approach to blood product purification; other more invasive and destructive techniques include use of aldehydes and ultraviolet light have proved too denaturing or destructive of the protein. Aside from blood products and blood product compositions, any protein solution having the possibility of viral contamination can be purified using the methods of the invention. For example, protein-containing solutions comprising mammalian milk, ascites fluid, saliva, placental extracts, tissue culture extracts, products of fermentation, transgenic derived products and recombinant proteins can all be purified by these methods. In Applicants' methods the preferred protein solutions for purification are blood products and blood product compositions.
In one embodiment of this invention, there is disclosed (1) a method for post-manufacture S/D treatment of human or animal derived proteins after size exclusion filtration of the protein; (2) a method for such S/D treatment that therefore uses much less solvent and detergent than previously used in the industry; and (3) means for removing the S/D by using silica beads in which the pore volume is filled with a three-dimensional cross-linked hydrophobic acrylic polymer to reduce protein binding to the silica. The use of the latter material allows the removal of detergent and reduces the endotoxin load in the product. The beads use the silica's natural ability to capture S/D while the polymer allows for greater than 95% recovery of the protein of interest, e.g. IgG.
It is disclosed herein the kinetics of viral inactivation in a protein as a post manufacturing step, specifically, in a purified immunoglobulin after fractionation and nanofiltration. We determined the amount of solvent and detergent could be reduced and still maintain a robust viral inactivation. The ability to decrease the amount of TNBP and Triton X-100 could reduce the amount of material required to remove the S/D to the point where it would be economically feasible to simply discard the sorbent, eliminating the need to regenerate the material. This would eliminate the requirement to validate sorbent regeneration, and minimize concerns about breakthrough of the S/D chemicals or extractables leaching from the material after repeated use.
In a preferred embodiment of the invention, the viral inactivation methods are performed on the human immune gamma globulin known commercially as RhoGAM® Ultra Filtered. (Ortho-Clinical Diagnostics, Inc., Raritan N.J.) Rho(D) Immune Globulin (Human) was the first successful prophylactic use of specific antibody to achieve antibody mediated immune suppression. RhoGAM® is an IgG immunoglobulin solution containing anti-Rho(D) at a dose of 300 micrograms of anti-D activity per dose. RhoGAM® can be given to the nonimmunized, Rho(D) negative pregnant woman at the appropriate time prevent future disease in her Rho(D) positive offspring. The disease is called hemolytic disease of the newborn or more specifically, Rh-erythroblastosis fetalis.
A smaller dose of anti-Rho(D), MICRhoGAM® Rho(D) Immune Globulin (Human) Micro-Dose (50 micrograms of anti-Rho(D)) is also sold by the Assignee hereof for treatment of women who have abortions and miscarriages at twelve weeks gestation or earlier. While the full dose protects the recipient for up to 15 ml of Rho(D) positive red cells, the smaller dose provides protection up to 2.5 ml of Rho(D) positive red cells. RhoGAM® is used as antenatal prophylaxis at 26 to 28 weeks gestation. Other indications include threatened abortion at any stage of gestation with continuation of pregnancy, abortion or termination of pregnancy at or beyond 13 weeks gestation, abdominal trauma or genetic amniocentesis, chorionic villus sampling (CVS) and percutaneous umbilical blood sampling (PUBS).
Most immunoglobulin injectable materials approved for use by the FDA and Bureau of Biologics have been produced by the alcohol fractionation procedure developed by Dr. E. Cohn of Harvard during the 1940s and described in Cohn et al., J. Am. Chem. Soc. 68, 459 (1946), incorporated herein by reference. This procedure, coupled with the careful selection of plasma negative for hepatitis infectivity, HIV, and other blood-borne pathogens determined by the most sensitive tests available, has insured that the resultant preparation of this procedure as safe. This fact can easily be demonstrated by the millions of non-infected recipients of product.
According to the current RhoGAM® Ultra Filtered manufacturing process, anti-D-containing plasma is fractionated (See Cohn et al., supra) and the resulting precipitate is resuspended in buffer and virally cleared using the Viresolve™ ultra-filtration membrane. The virally-cleared material is diafiltered and concentrated using a Biomax size exclusion filter. Protein concentration and pH are adjusted and the resulting bulk material is filled into syringes. See commonly-assigned U.S. Pat. No. 6,096,872.
Solvent/detergent treatment is widely accepted as a method for inactivating lipid-enveloped viruses in plasma and plasma-derived therapeutic proteins. Numerous studies have demonstrated the effectiveness of this process with plasma, immunoglobulin preparations, coagulation factor concentrates and other plasma proteins.
Typically, when performing a solvent/detergent treatment, solvent and detergent are added to plasma at the start (front end) of a manufacturing process at concentrations of 1% each, or at an intermediate step in processing at concentrations of 0.3% and 1.0% respectively. The instant invention discloses a unique viral inactivation step in that lower concentrations of solvent (ranging from about 0.003%-less than 0.3% TNBP) and detergent (ranging from about 0.01% to less than 1.0% Triton X-100) are used, post manufacture, to inactivate virally-cleared, lipid-free bulk product. The removal process for eliminating solvent and detergent from the final product is also unique in that it is accomplished without an extraction step. Instead solvent and detergent are removed directly by use of a silica bead sorbent material. While the sorbent material can be regenerated, it is preferred that the sorbent material will be for one-time use only.
As herein disclosed it is preferable to add a virus-inactivation step post manufacture of the current RhoGAM® Ultra Filtered process. The prior art has considered concentrations of 1.0% detergent and 0.3% tri-n-butyl phosphate (TNBP) solvent necessary for robust removal of viral activity. In contrast to these high concentrations of S/D, using the methods of the instant invention, human Immune gamma globulin (RhoGAM®) bulk material can be treated post manufacture with about 0.01%-less than 1.0% detergent (such as Triton X-100) and about 0.003% to less than 0.3% solvent (such as Tri (n-butyl) phosphate (TNBP)). The treatment in a preferred embodiment is for a minimum of about 1 hour at 15° C.-25° C. The above ranges for solvent and detergent will be expected to vary with variations in temperature and/or extended times of incubation; for instance, increased temperatures and/or extended incubation times will allow for even lower S/D concentrations. After treatment, solvent and detergent are preferably removed by passage of material through a column containing a silica sorbent material, for example, SDR Hyper D Solvent-Detergent Removal sorbent (manufactured by the BioSepra Division of Ciphergen BioSystems, Inc., Fremont, Calif.). The sorbent is composed of silica beads in which the pore volume is filled with a three dimensional cross-linked hydrophobic polymer that retains solvent and detergent. Virus inactivated RhoGAM® (RhoGAM SD™) is collected, diafiltered and concentrated using a Biomax filter. Polysorbate 80 concentration, pH and protein concentration may then be adjusted such that the final RhoGAM SD™ product is consistent with the current formulation.
The S/D step may also be employed at the front end of the manufacture process. Where the S/D step is employed in the inventive process at the front end of the manufacture, it is preferable to employ about 0.2% to less than 1.0% solvent and about 0.2% to less than 1.0% detergent.
A flow chart of the proposed manufacturing steps required for viral inactivation is provided in FIG. 1